使用新型溶酶体运输探针鉴定控制溶酶体稳态的因子。,Scientific Reports

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使用新型溶酶体运输探针鉴定控制溶酶体稳态的因子。
Scientific Reports ( IF 3.8 ) Pub Date : 2019-08-12 , DOI: 10.1038/s41598-019-48131-2
Shunsuke Ishii ₁ , Akira Matsuura ₂ , Eisuke Itakura ₂

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溶酶体在很大程度上通过分泌途径，自噬和内吞作用导致细胞内和细胞外蛋白质的显着降解。因此，溶酶体稳态的失调影响多种细胞功能。但是，尚未开发出一种直接且定量的方法来测量溶酶体途径的完整性。在这里，我们报告基于质粒的溶酶体-METRIQ（通过RatIo定量测定蛋白运输完整性）探针，该探针能够通过流式细胞仪通过溶酶体绿色和胞质红色荧光蛋白对溶酶体完整性进行简单定量。在培养的细胞中，溶酶体-METRIQ探针不仅检测到了溶酶体途径的抑制，而且还检测了溶酶体活性（如溶酶体生物发生）的上调。为了确定溶酶体稳态的相关因素，我们进行了化合物筛选，发现细胞周期蛋白依赖性激酶（CDK）抑制剂kenpaullone和purvalanol A诱导组织蛋白酶D的合成和溶酶体数量的增加。随后的研究表明，CDK5维持溶酶体稳态，而与细胞周期停滞无关。我们的结果表明，溶酶体-METRIQ探针是一种有效的工具，可用于测量哺乳动物细胞中的溶酶体活性。

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Identification of a factor controlling lysosomal homeostasis using a novel lysosomal trafficking probe.

Lysosomes are largely responsible for significant degradation of intracellular and extracellular proteins via the secretory pathway, autophagy, and endocytosis. Therefore, dysregulation of lysosomal homeostasis influences diverse cellular functions. However, a straightforward and quantitative method to measure the integrity of the lysosomal pathway has not been developed. Here, we report the plasmid-based lysosomal-METRIQ (MEasurement of protein Transporting integrity by RatIo Quantification) probe that enables simple quantification of lysosomal integrity by lysosomal green and cytosolic red fluorescent proteins using a flow cytometer. In cultured cells, the lysosomal-METRIQ probe detected not only suppression of the lysosomal pathway but also upregulation of lysosomal activity such as lysosomal biogenesis. To identify factors involved in lysosomal homeostasis, we carried out compound screening and found that the cyclin-dependent kinase (CDK) inhibitors kenpaullone and purvalanol A induce synthesis of cathepsin D and an increase in the number of lysosomes. Subsequent studies revealed that CDK5 maintains lysosomal homeostasis independently of cell cycle arrest. Our results suggest that the lysosomal-METRIQ probe is an effective and efficient tool for measuring lysosomal activity in mammalian cells.

更新日期：2019-08-12

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